Figure 3
From: Mycobacterium bovis BCG promotes tumor cell survival from tumor necrosis factor-α-induced apoptosis
BCG-mediated SHH signaling induce expression of COP1. (A and B) The kinetics of SHH signaling activation was assayed by quantitative real time RT-PCR for expression of SHH signaling markers SHH, GLI1, GLI2, PTCH1 and SMO (A) and by immunoblotting for SHH, NUMB and pGSK-3β (B). (C-F) SHH signaling was inhibited using specific pharmacological inhibitors, Cyclopamine (SMO inhibitor) and Betulinic Acid (GLI inhibitor). Validation of inhibitors used (C) and analysis the expression of COP1 by quantitative real time RT-PCR (D), luciferase assay (E) and immunoblotting (F). *p < 0.05 (one-way ANOVA), as compared to DMSO treated cells. (G-I) A549 cells were transfected with SHH specific siRNA to assess COP1 and p53 protein levels after indicated treatment (G and H). Validation of SHH siRNA (I). (J) A549 cells overexpressing SHH expression construct were analyzed for COP1 transcripts and COP1 promoter activity. *p < 0.05 (t-test), as compared to pcDNA3 transfected cells (K) A549 cells were infected with BCG and the recruitment of GLI1 transcription factor at COP1 promoter upon infection with BCG in A549 cells was evaluated by ChIP assay. *p < 0.05 (one-way ANOVA). Data is representative of mean ± SEM, n = 3 and all blots are representative of 3 independent experiments. Med, Medium; NT, non-targeting.